Compound 21 was separated as white substance. The polar compound gave an uncommon green colour reaction with anisaldehyde/sulphuric acid, and EI MS indi-cated a molecular ion at 168 Dalton. The search in AntiBase with the mass delivered 27 hits and the search in the MS Nist Database gave 3 hits. The proton NMR spec-trum indicated in the aromatic range two broad signals at 10.51 and 8.56 attributed to two exchangeable protons, two doublets at 6.58 and 6.53 and one doublet of doublet at 6.46 for a 1,2,4-trisubstituted benzene moiety. In the aliphatic range one
singlet at 3.77 can be assigned to a methylene group. The substructure search in AntiBase with the 1H NMR spectrum and the mass indicated 3 hits.
Figure 14: 1H NMR spectrum (300 MHz) of homogentisic acid (21) in DMSO-d6
Comparison of the proton NMR spectrum with theses hits matched with ho-mogentisic acid (21).
Homogentisic acid is a derivative of the gentisic acid (22) and is formed as in-termediate in phenylalanine or tyrosine metabolism. [63] It is reported that the pres-ence of homogentisic acid (21) in the urine is a symptom of the illness Alkaptonuria (black urine disease), a rare inherited genetic disorder of tyrosine metabolism. [64]
O OH O
H
OH
1 1´
3´ 5´
OH O
H
OH O
1´
3´ 5´
21 22
Obtained as white powder, compound 23 indicated in the EI MS a molecular ion at m/z 182. Its high-resolution delivered the molecular formula C9H10O4. The ∆m = 14 between 23 and 21 was attributed to a methyl group and suggested that compound 23 is just the methyl ester of 21, i.e. methyl homogentisate. It was first obtained by hydrolysis with methanol in hydrochloric acid from Phaseoloidin, which was isolated from the seeds of the plant Entada phaseoloides. [65]
Pseudoalteromonas Strain T 268 33
OH O
H
O O
23
Homogentisic acid (21) exhibited phytotoxic activities on the root of the mono-cotyledonous and dimono-cotyledonous seeds with 333 μg/ml and 67 μg/ml, respectively.
5.1.3 Euphamycin A
The electron impact (EI) mass spectrum showed a molecular peak at m/z 306 for the light pale yellow oily compound 25. The HREIMS delivered the molecular for-mula C11H14O4S3. The 1H NMR spectrum displayed one broad signal at 8.38, one singlet at 3.86 with an intensity of two protons and three other singlets integrating for three protons each at 2.36, 2.32 and 2.25 suggesting that they may be aromatic methyls or S-methyls, as the chemical shift of O-methyls is in the range of 3.00 and 4.00.
Figure 15: 1H NMR spectrum (600 MHz) of euphamycin A (25) in DMSO-d6
The 13C NMR spectrum of compound 25 exhibited one carbonyl carbon at 172.2, six quaternary carbons at 127.8, 126.6, and 124.5 (2 C) including two car-bons bearing oxygen at 151.8 and 149.6. Additionally, one methylene group at 35.2 and three methyl signals at 18.8, 18.6 and 17.6 were observed.
Figure 16: 13C NMR spectrum (125 MHz) of compound 25 in DMSO-d6
The search in AntiBase, the Dictionary of Natural Product and the Chemical Abstracts by considering the NMR data, molecular mass and formula gave no hits, suggesting compound 25 as a new metabolite. A second search using only NMR sub-structures delivered similarities with compound T-1801-A (24), a compound isolated from Pseudomonas sp. sc-1801.[66]
S
S O S H
OH
24
The 2D NMR experiments, namely HSQC, HMBC and the 1D NOESY were measured. The HMBC spectrum indicated a correlation of the methylene singlet at 3.86 to the carbonyl at 172.2, the oxygenated sp2 at 149.6 as well as to the qua-ternary carbons at 127.8 and 124. 6 and gave fragment I.
O H
OH O
172.2
149.6 127.8 124.6 35.2
Figure 17: HMBC correlations in fragment I of compound 25
Pseudoalteromonas Strain T 268 35
Comparison of data with those of compound T-1801-A (24) confirmed the chemical shifts of the methyl carbons as S-methyls; they showed individual long range correlations to 124.6, 126.6 and 124.6, respectively.
S
124.6 17.6
S
126.6 18.6
S
124.6 18.8
Figure 18: S-methyl fragments of compound 25
The connectivity between the four fragments was difficult to find out. The inter-pretation of the 1D-NOESY delivered the following evidences: irradiation of the methylene group at 3.86 delivered only one weak NOE effect with the thiomethyl signal at 2.25, so that obviously only one SMe group is in o-position to the methyl-ene group; the other o-position must be occupied by OH. Similarly, irradiation of the methyl signal at 2.25 increased slightly the methylene signal, however, did not in-fluence any of the other two SMe signals: It follows, that an OH group must be next to this SMe group, so that 25 is the only possible structure.
Figure 19: 1D-NOESY spectrum (600 MHz) of the proton at 3.86 in DMSO-d6
This information, in addition to the comparison with data from T-1801-A (24) suggested the structure 25 of a tris-thiomethyl-homogentisic acid, for which the name euphamycin A was given. Like the hydroquinone T-1801-A (24), which is re-ported to have antimicrobial and antifungal activities, euphamycin A (25) showed activities against Bacillus subtilis, Bacillus brevis, Micrococcus luteus, Escherichia coli, Paecilomyces variotii and Phytophthora infestans. As for the antitumour
3.86
Weak NOE
otic resorthiomycin [67] (26), it showed also cytotoxic activities against L1210, Jurkat, MDA-MB-321, MCF-7, and Colo-320 cells (Table 26).
S
The temperature and solvent sensitive compound 27 was isolated as pale oil. It showed a UV absorbing spot at 254 nm with gave a yellow colour after spraying with anisaldehyde/sulphuric acid. The EI mass spectrum showed a molecular peak at m/z 320 and its high-resolution delivered the molecular formula C12H16O4S3. The 1H NMR spectrum was very similar to that of compound 27 with the methylene singlet at 3.86 and the three S-methyls at 2.36, 2.32, 2.25. The major difference was the additional singlet of three protons at 3.61 attributed to a methoxy group.
The molecular mass of compound 27 in EI spectrum indicated a mass 14 unit higher than that of compound 25 confirming the presence of a methoxy group as in-dicated in the 1H NMR spectrum. No hits were found in an AntiBase search, conse-quently compound 27 was deduced as the methoxy derivative of compound 25; it is also a new natural product, which was named euphamycin B. Due to the small amount and the high sensitivity of the compound, the 2D NMR spectra could not be measured. Euphamycin B showed activity against Gram-positive bacteria.
S
Strain T48 37
5.2 Strain T48
The TLC of strain T48 extracts indicated distinctive UV active zones, which showed multiple colour reactions with anisaldehyde/sulphuric acid. The biological screening of the crude extract showed antimicrobial activity against different test organisms, and indicated cytotoxicity.
Crude extract -acetic acid methyl ester
Genistin
(4-Hydroxy-phenyl) -acetic acid methyl ester
Indol-3-yl-acetic acid
Indol-3-yl-acetic acid methyl ester
Figure 20: Work-up scheme of the strain T48A 5.2.1 Genistin
Compound 28 was obtained as white to light yellow powder, which was easily soluble in DMSO. ESI MS showed a quasimolecular ion at m/z 431 [M-H]-. The pro-ton NMR spectrum of the compound measured in DMSO-d6 exhibited in the aro-matic region nine protons among which two were exchangeable and appeared at 12.82 for a chelated proton and 9.51, one singlet at 8.41, two doublets at 7.41 (J
= 8.8 Hz) and 6.82 (J = 8.7 Hz) each with the intensity of two protons attributed to a para disubstituted benzene ring. In addition, two protons in meta position at 6.72 (d, J = 1.3 Hz) and 6.45 (d, J = 1.3 Hz) were seen. The aliphatic region displayed a set of protons due to a sugar moiety in the range of 3.75-3.18, and the anomeric proton signal appeared at 5.10 (d, J = 1.8 Hz). Two exchangeable protons at 5.38 and 4.58 were also visible.
Figure 21: 1H NMR spectrum (300 MHz) of genistin (28) in DMSO-d6
The 13C NMR spectrum of the compound displayed 21 carbons signals, which were sorted as seven quaternary carbon signals including one carbonyl at 180.5, seven sp2 methines, five sp3 methines and one methylene. From the 13C NMR data two aromatic hydroxyl groups ( 162.9, 157.4) and two other aromatic sp2 carbon attach to the oxygen ( 161.6, 157.2) can be derived.
Figure 22: 13C NMR spectrum (125 MHz) of genistin (28)
The H,H COSY spectrum confirmed the presence of meta and para disubsti-tuted aromatic systems suggested by the 1H NMR spectrum.
Strain T48 39
Figure 23: Fragments in genistin (28)
The search in AntiBase, [68] the Dictionary of Natural Products and the Chemical Abstracts, [69] with the mass and the molecular formula delivered three proposals, and compound 28 matched with the data of the isoflavone genistin. This glycoside of genisteine is very predominant in plants. Genistein-7-glucoside is known to stimulate estrogen dependent breast cancer cell growth in vivo. [70] Compound 28 might be new from bacteria; however, it is more plausible that it came from the malt extract of the nutrient broth.